3. TRD#2: In vitro molecular microscopy pipeline Abstract Over the next five years we will continue our mission of building a complete pipeline for what we refer to as ?in vitro molecular microscopy?. We will undertake a very thorough investigation of the value and performance of a variety of high-end microscope accessories that are available (counting direct detectors, Cs correctors, energy filters, phase plates) and fully integrate the phase plate and Cs corrector into the Leginon system. The phase plate will be critically important to the success of several of our driving biological projects. We aim to more completely understand and optimize the performance of the high-end vs. mid-range microscopes and the value of each of the various accessories. This is especially important due to the very high demand and long waiting lists for access to the most expensive instruments. We are also committed to support ?high-throughput? negative stain screening. Spotition will transfer up to 96 negatively stained samples onto a single grid and each of these samples needs to be automatically imaged and ideally semi-automatically analyzed as rapidly and efficiently as possible. We will build specialized applications in Leginon and Appion to support this pipeline driven by several important Driving Biological Projects. We will also continue to expand Appion by adding new features, programs and applications into the pipeline, add functionality for remote web-based Leginon control, and explore options for supporting cloud-based computing for users who may not have access to local high end computational infrastructure. These advances will enable a model where users mail samples to NRAMM and perform both data collection and processing remotely.